Extremely low frequency magnetic fields (ELF-MFs) are classified as a possible carcinogenic factor (Group 2B). This study assessed the association between ELF-MFs and childhood cancer through a systematic review and meta-analysis.Three databases were searched in January 2020. We conducted a meta-analysis for the association between the ELF-MFs exposure level and childhood cancer.A total of 33 studies were identified. Thirty studies with 186,223 participants were included in the meta-analysis. Children exposed to 0.2-, 0.3-, and 0.4-μT ELF-MFs had a 1.26 , 1.22 (95% CI 0.93–1.61), and 1.72 (95% CI 1.25–2.35) times higher odds of childhood leukemia. In childhood brain tumors, children exposed to 0.2-μT had a 0.95 (95% CI 0.59–1.56) times higher odds, and those exposed to 0.4-μT ELF-MFs had a 1.25 (95% CI 0.93–1.61). Children exposed to 0.2- and 0.4-μT ELF-MFs had a 1.10 (95% CI 0.70–1.75) and 2.01 (95% CI 0.89–4.52) times higher odds of any childhood cancers.Significant associations were observed between exposure to ELF-MFs and childhood leukemia. Furthermore, a possible dose-response effect was also observed. The debate on the effect of electromagnetic fields (EMFs) on the human body still continues, and several studies have investigated the effect of magnetic fields that are not well shielded by objects –3. The qThe WHO International Advisory Committee presented a Research Agenda for ELF-MFs whereby they indicated that epidemiological studies are of primary importance in health risk assessment, and that high-priority research needs to conduct pooled analyses of existing childhood cancer studies . Until 2The first epidemiological study of the association between the exposure to ELF-MFs and childhood cancer was published in 1979 . Over thTherefore, it is necessary to comprehensively search and review the literature with an appropriate methodology, and to carry out a meta-analysis to confirm the association between exposure to ELF-MFs and childhood cancer.We aimed to assess the association between ELF-MFs and childhood cancer through a systematic review and meta-analysis of relevant studies.The procedure adopted in this study followed the guidelines of the Cochrane Collaboration . MoreoveUsing the search strategy recommended by Cochrane , the seaStudies were searched, reviewed, and selected by two authors independently (J. P. and G. S.), using predefined criteria. Full texts for studies that met the inclusion criteria were identified and reviewed. In cases where no consensus could be reached, the issue was resolved through either further discussion or a separate examination by another author (J.L.), who is a statistical expert. However, there were no notable inconsistencies with the initial selection.The inclusion criteria were: (1) epidemiologic studies (cohort or case-control design); (2) studies on exposure of ELF-MF; (3) studies with childhood cancer; (4) studies indicating subject number by each group exposure level; (5) based on magnetic field measurements or the calculated field; and (6) the article written in English. Abstracts presented at congresses, reviews, letters, editorials, and unpublished data were excluded.The Newcastle–Ottawa Scale (NOS) was used to determine the risk of bias in the studies that were included  in the sJ.P. and G.S. independently extracted the relevant data from each study, and discussed and resolved any issues that emerged. The data that were abstracted included information on: the first author, publication year, study design, country of origin, eligible population size, study population characteristics, registry group, exposure measurement, and matching variables.I2 statistic [p-value of less than 0.1 was used as an indicator of the presence of heterogeneity, the I2 statistic provides a measure for quantifying the heterogeneity with regard to the proportion of between-study variation based on the total variation in the estimates. In this study, an I2 value of greater than 50% was considered to denote the existence of substantial heterogeneity among the study results. We conducted analyses that were stratified by the level of exposure of EMF, and further evaluated the dose–response effect of EMF exposure. Moreover, the sensitivity analysis was performed by individually excluding each study to assess its influence of each study on the overall result of the meta-analysis.We analyzed the data by using Review Manager (version 5.3) for the association between exposure to EMF and childhood cancer. Meta-analyses of the risk of childhood cancer outcomes were conducted to obtain pooled odds ratios (ORs) with the 95% confidence intervals (CIs) in a random effects model in case-control studies. The cohort studies were excluded in meta-analysis due to its differences in EMF exposure levels and outcome variables between studies. The heterogeneity among studies was estimated by using the tatistic , 32 as wp-value of less than 0.05 was considered to be statistically significant.The publication bias was examined graphically by using a funnel plot of a study’s effect size against the standard error. A two-tailed Our systematic search found a total of 3,933 studies. After removing duplicate articles, a total of 3,250 studies were excluded for lack of eligibility for study inclusion on a review of the study title and abstract. A full-text review of the remaining 186 studies was undertaken, and a further 153 studies were excluded. Finally, 33 studies were included in the systematic review for the association of ELF-MFs and childhood cancer , 33–56, The included three cohort studies were unable to perform meta-analysis due to differences in exposure levels and outcomes between studies. In a cohort study investigating the risk of cancer in children living close to overhead power lines with a magnetic field of 0.01-uT, the standardized incidence ratio was 0.97 , with no statistically significant increases in all cancers . Two of The study characteristics included the year of diagnosis and the registry group . The metThe absolute numbers of childhood cancer cases and controls for the exposure level of the ELF-MFs were presented in The risk of bias in the studies was evaluated by the NOS for all 30 articles included in the meta-analysis. A total of 30 articles that met all of the selection criteria were assessed for their quality. The results associated with the quality ratings of the retrieved studies are shown in We identified 30 studies with 186,233 subjects that report results on the association between ELF-MFs and childhood cancer. Meta-analyses were conducted for pediatric leukemia, brain tumors, and any childhood cancers by level of exposure to ELF-MFS.2 = 0%). For the 0.3-μT exposure level of ELF-MFs, the pooled summary OR was 1.22 (0.93–1.61) without any significant heterogeneity , and there was evidence of publication bias (The results of six study were used to examine an association of exposure to ELF-MFs with any childhood cancers. The pooled summary OR of the 0.2-μT exposure level of ELF-MFs was 1.10 (0.70–1.75) , whereasion bias .The sensitivity analysis was performed after excluding either studies that used calculated fields measurement method or studies that identified very small cases even though many participants were examined and, hence, affected to pooled estimate of meta-analysis significantly , 22. TheWe conducted a meta-analysis on each type of cancer to derive the large-scale integrated values for the association between ELF-MFs exposure and childhood cancer among more than 85,000 children diagnosed with childhood cancer in 15 countries. In this study, based on the systematic review methodology, all articles that presented pediatric cancer, including leukemia, lymphoma, and brain tumor, were included in a comprehensive search. Pooled analysis studies have been conducted on the association between childhood cancer and ELF-MFs exposure , 59. HowThe existence of between-study heterogeneity can generate a statistical problem when attempting to interpret the results of the meta-analysis. Ideally, the studies, whose results are being combined in the meta-analysis, should all be undertaken in the same way and with the same experimental protocols: study heterogeneity is a term used to indicate that this ideal is not fully met . Meta-an2 = 0%) as well as an absence of any evidence for publication bias. In addition, this study derived a large-scale integrated value using larger number of included studies than previous studies. Our results showed slightly higher pooled estimates than previous results, which are 1.07–1.08, 1.11–1.16, and 1.44–2.00 times higher odds for exposures to 0.2-, 0.3-, and 0.4-μT, respectively [This study showed that children exposed to 0.2-μT ELF-MFs had a 1.26 (95% CI 1.06–1.49) times higher odds of child leukemia, those exposed to 0.3-μT had a 1.22 (0.93–1.61) times higher odds, and those exposed to 0.4-μT had a 1.72 (1.25–2.35) times higher odds. These results are based on statistical homogeneity  times higher odds of childhood brain tumors, and children exposed to 0.4-μT ELF-MFs had a 1.25 (0.93–1.61) times higher odds. However, the estimates were not statistically significant. Based on previous pooled analysis, studies showed a 0.95 times on exposure to 0.2-μT and 1.14-times higher odds on exposure to 0.4-μT ELF-MFs, and similar results were found in this study [According to the pooled OR value of four studies, children exposed to 0.2-μT ELF-MFs had a 1.10 (0.70–1.75), and those exposed to 0.4-μT ELF-MFs had a 2.01 (0.89–4.52) times higher odds of any childhood cancer, However, it did not show a high-intensity relation. There have been no pooled analysis studies of any childhood cancer conducted thus far.Public concern about EMFs is considerably high, and many countries have implemented a lot of policies related to EMFs , 62. AnyAlthough we identified three cohort studies that met the study eligibility criteria, these studies were not able to include in the meta-analysis. In the cohort study, the incident ratio of childhood leukemia was 1.6 and any childhood cancer was 1.5 in the group exposed to 0.2-μT, however, it was not statistically significant . In the The ELF-MFs measurement variation was not considered in our meta-analysis. Although some studies used a long-term measurement, others used a spot measurement; however, no distinction has been made in our meta-analysis because of the limited number of studies. Nonetheless, our pooled results showed a non-significant heterogeneity among studies.Another limitation is that all studies included in our study were case-control studies, which reduces the strength of the obtained results because case–control studies are subject to selection bias and other methodological problems. However, all studies that were included in this meta-analysis were the matched case-control studies except one, which had the case and control groups from the same registry. Therefore, it can be seen that the bias shown in case-control studies has diminished.In this large pooled analysis of more than 36,000 children diagnosed with childhood leukemia, statistically significant associations were observed between exposure to ELF-MF and childhood leukemia. Furthermore, the intensity of the association between exposure to ELF-MFs and childhood leukemia was high, as indicated by the dose–response effect.The risk of ELF-MFs, which have been classified as a possibly carcinogenic (Group 2B) factor based on limited evidence in humans, can be ascertained through precise evidence from the integrated results of this study.S1 TableSearch strategies and search results for each database .(DOCX)Click here for additional data file.S1 Checklist(DOC)Click here for additional data file.


In CoCr, the low adhesion effect with low frictional coefficient may result in excessive taper-slip, especially with the use of moderate viscosity bone cement.Although some reports suggest that taper-slip cemented stems may be associated with a higher periprosthetic femoral fractures rate than composite-beam cemented stems, few studies have focused on the biomaterial effect of the polished material on the stem–cement interface. The purpose of this study was to investigate the relationship between surface roughness of materials and bone cement. Four types of metal discs—cobalt-chromium-molybdenum alloy (CoCr), stainless steel alloy 316 (SUS), and two titanium alloys (Ti-6Al-4V and Ti-15Mo-5Zr-3Al)—were prepared. Five discs of each material were produced with varying degrees of surface roughness. In order to evaluate surface wettability, the contact angle was measured using the sessile drop method. A pin was made using two bone cements and the frictional coefficient was assessed with a pin-on-disc test. The contact angle of each metal increased with decreasing surface roughness and the surface wettability of metal decreased with higher degrees of polishing. With a surface roughness of Ra = 0.06 μm and moderate viscosity bone cement, the frictional coefficient was significantly lower in CoCr than in SUS ( The introduction of Charnley’s low friction arthroplasty was a technological revolution and has become the most successful total hip arthroplasty (THA) . CharnlePeriprosthetic femoral fractures (PPFs) following THA are devastating complications that are associated with functional limitations and increased overall mortality . The NorFour types of metal discs—cobalt-chromium-molybdenum alloy (CoCr), stainless steel alloy 316 (SUS), and two titanium alloys (Ti-6Al-4V and Ti-15Mo-5Zr-3Al)—were prepared . The shaSurface wettability is one of the characteristics of a metal surface and indicates whether the metal has a water-shedding or water-attracting surface ,23. For The contact angle measurements were carried out using the sessile drop method with an optical contact angles meter . Saline was drawn into a micrometric syringe and dropped onto a metal disc (1.0 μL). After 28 s, the droplet was captured using a camera . Temperature (22–23 °C) and relative humidity (50–55%) were maintained during all analyses. Then, the captured image was analysed using FTA 32 software  . The conFor the evaluation of a frictional coefficient, a pin-on-disc test was performed. At first, a pin was made by two bone cements  and Palacos R ) using vacuum mixing. Endurance, in which the viscosity is similar to that of Smart Set MV , represented a moderate viscosity bone cement, whereas Palacos R represented a high viscosity bone cement. After the hardening of the bone cement, the pin was polished using a lathe and #400 sandpaper. The pin was attached to the pin-on-disc tribometer  and the contact surface was polished further using #800–#2000 sandpapers and #2000 emery paper, with 20 rpm of 1N. The shape of the pin was 5 mm in diameter and 45 mm in length .The tribological experiments were performed using the pin-on-disc tribometer , according to the report by Fukui et al. . The disp < 0.05. All analyses were performed using SAS .Using a generalised linear mixed effects model with surface roughness as a random effect, a quadratic curve was drawn using plots between the surface roughness and frictional coefficient in each metal. Then, the corrected values of the surface roughness were calculated with adjustments within 0.03 and the actual measured or corrected values were identified for comparison with even surface roughness. In order to compare the surface roughness between groups, a generalised linear mixed effects model with surface roughness was employed again. Statistical significance was set at The relationship between the contact angle and surface roughness is shown in The relationship between the frictional coefficient and surface roughness is shown in p = 0.0073). By using Endurance and Palacos R with a surface roughness of Ra = 0.1 μm, the frictional coefficients in Ti-6Al-4V and Ti-15Mo-5Zr-3Al were significantly lower than that in CoCr (p < 0.0001). Using Endurance and Palacos R with a surface roughness of Ra = 0.4 μm, the frictional coefficient in Ti-15Mo-5Zr-3Al was significantly higher than in Ti-6Al-4V (p < 0.0001). With a surface roughness of Ra = 0.4 μm, the frictional coefficient in Ti-6Al-4V was significantly lower than in CoCr and SUS, whereas there was no significant difference between Ti-15Mo-5Zr-3Al and CoCr and SUS.The comparison of frictional coefficients in the metal samples is shown in Types of cemented stem fixation are classified as composite-beam design and taper-slip design . ImplantCommonly, there are three types of materials that are used for cemented stems—CoCr alloy, SUS, and titanium alloy. In polished stems, stems made of titanium alloy are not favoured because they are associated with early failure and two disadvantages: a stiffness of about 50% of that of CoCr alloy or SUS and a susceptibility of the material to crevice corrosion . ConversClinically, there is a difference among taper-slip stems in terms of the incidence of PPF. According to the National Joint Registry of England, Wales, and Northern Ireland database on 257,202 THAs, analysis of the four cemented femoral stem brands showed a PPF incidence of 0.12% in Exeter V40, 0.07% in the Charnley stem , 0.46% in the CPT stem, and 0.14% in the C-stem . The incSome limitations of our study must be noted. First, the sample size was small. Although statistically significant data were acquired, the Ra parameters that could be compared were limited. Ideally, more samples should have been included. Second, the frictional coefficient between the metal and bone cement is dependent on the environment, that is, whether the condition is wet or dry . In thisThe surface wettability of a metal decreases with increased polishing and CoCr had the lowest surface wettability compared to SUS with a surface roughness of Ra = 0.06 μm. Furthermore, the frictional coefficient of CoCr was lower compared to that of SUS. In CoCr, the low adhesion effect with low frictional coefficient may result in excessive taper-slip, especially when using moderate viscosity bone cement. However, further multifaceted studies that include biomechanical assessments are required to confirm our results.


O-robinoside-7-O-rhamnoside) was isolated from the aerial parts of Astragalus falcatus Lam. The monotherapy with robinin was not efficient in alleviating symptoms of AA. The combination of MTX with robinin was similarly active as MTX alone in reducing the hind paw volume and change of body weight during the whole experiment. The combination, however, reduced plasma levels of Interleukin-17Aand activity of gamma-glutamyl transferase in joint more efficiently then MTX alone. Our results demonstrate that the novel combination of robinin and MTX mildly improved the reduction of inflammation in experimental arthritis.Anti-inflammatory potential of orally administrated bioflavonoid-robinin, active sub-stance of original drug Flaroninum™ (FL), was investigated in the combination with methotrexate (MTX) and in monotherapy in rats suffering from adjuvant-induced arthritis (AA). Robinin (kaempferol-3- Rheumatoid arthritis (RA) is an autoimmune inflammatory disease, which preferably targets the synovial joints and is associated with premature mortality and socioeconomic burdens . The patAstragalus which is broadly distributed throughout the temperate and arid regions and known to contain phytochemicals, which are found in most parts of the plant and have been associated with multiple actions i.e., anti-inflammatory, pain relieving and protective effects /[Day 1]) × 100 − 100 = value [%]. The body weight of the animals was measured daily. The changes in body weight are shown as average of the weight gain [g]. Weight measured on the day  minus weight measured on day 1. The mathematical formula we used is as follows: [n Day] − [Day 1] = value [g].2PO4, 50 mM of Na2HPO4, 68 mM of NaCl,15 mM of EDTA; pH 8.1) at 1:9 (w/v) by Ultra Turax TP 18/10 (Janke and Kunkel) for one min at 0 °C. The biochemical substrates (44 mM of methionine and 8.7 mM of L-γ-glutamyl-p-nitroanilide) were dissolved in isopropyl alcohol (65%) to final concentrations of 2.5 mM and 12.6 mM, respectively. After one hour incubation at 37 °C, the reaction was stopped by adding 2.3 mL of cold methanol. Tubes were centrifuged at 5000 rpm for 20 min (Centrifuge Eppendorf). Supernatant’s absorbance (product p-nitroaniline) was determined on spectrophotometer Specord 40 at 406 nm. Solution mix without or without substrate or acceptor were used as blanks.The cellular activity of γ-glutamyltransferase (GGT) was measured on day 28 in the spleen and hind paw joint tissue homogenates using the method of Orlowski and Meister  and modiA—absorbance;c—concentration of p-nitroaniline [µg/mL];k—coefficient from calibration curve A = k. c.Mathematical formula explanatory note:®; Waltham, MA, USA) according to the instruction of the manufacturer. Statistical analyses. The average values ± SEM were calculated. Significant differences between control animals, untreated animals, and treated groups of animals were determined by ANOVA. The post hoc test (Tukey-Kramer) was applied in cases where differences between groups was significant. The levels of significance after the post hoc screening were specified as followed: not significant (p > 0.05), significant (p ≤ 0.05), very significant (p ≤ 0.01), and highly significant (p ≤ 0.001).The level of plasmatic Interleukin-17A (IL-17A) was determined using an enzyme-linked immunosorbent assay type kit , IL-17A concentration were markedly enhanced at day 14 and then gradually dropped within the duration of the experiment and were only insignificantly higher at the end of the experiment. Only on day 14 in the MTX group did the plasma levels of this cytokine decrease significantly compared to AA group. The treatment of combination of RB-MTX group showed a significant decrease  in level50 is higher than >1 g/kg in mice [This is the first in vivo study, were the anti-inflammatory effect of the active substance robinin (RB) on the development of adjuvant arthritis (AA) was investigated. We have also examined the combined administration of RB with antirheumatic drug MTX. Adjuvant arthritis is recognized as one of extensively used animal model for studying the series of inflammatory processes that occur in RA and validating novel anti-inflammatory and anti-rheumatic drugs ,21,26,27 in mice ,35. AccoAA animals have a quick onset and progression of disease with poly-articular inflammation, which is manifested by increased volume of the hind paw and a typical course of loss of body weight .In all AA rats, the body weight gain was significantly lower than in control animals , which mSuppressing cytokines with natural products has become an important focus in the development of new drugs to treat RA . InflammIn our study, we examined the effect of RB on the plasma concentration of IL-17A during the development of AA. Recent works suggests that IL-17 contributes to RA chronicity and late pathogenic responses through synovial inflammation and hyperplasia .p-hydroxyphenylacetic acid. Formation of p-hydroxyphenylacetic acid was increased in animals that had previously received a diet containing kaempferol or robinin. The same metabolites were shown to be formed on incubation of robinin with the microflora [This also applies for AA ,63,64. Rcroflora . The 3-fcroflora . Kaempfecroflora . Moreovecroflora . ProbablIn this study, the anti-inflammatory and anti-arthritic activity of robinin (RB) was evaluated for the first time. RB in monotherapy was not efficient in reducing any of parameters measured. The beneficial properties of MTX monotherapy were not enhanced by the co-administration of RB on CBW and HPV. However, the combination of MTX-RB was more effective in decreasing the activity of GGT in the hind paw joint and in lowering the plasmatic level of IL-17A than MTX alone. However, further investigations on the mechanism of anti-inflammatory and anti-arthritic action, as well as the oral pharmacokinetic profile of RB, are needed for better understanding of robinin efficacy in RA. Importantly, RB is a very safe compound and, hence, potentially suitable for combination regimens.


Heterodera glycines, the most economically damaging parasite of soybean [Glycine max L. (Merr)], has been reported in previous research. However, the family of genes encoding FLPs of H. glycines were not identified and functionally characterized. In this study, an FLP encoding gene Hg-flp-22 was cloned from H. glycines, and its functional characterization was uncovered by using in vitro RNA interference and application of synthetic peptides. Bioinformatics analysis showed that flp-22 is widely expressed in multiple nematode species, where they encode the highly conserved KWMRFamide motifs. Quantitative real-time (qRT)-PCR results revealed that Hg-flp-22 was highly expressed in the infective second-stage juveniles (J2s) and adult males. Silencing of Hg-flp-22 resulted in the reduced movement of J2s to the host root and reduced penetration ability, as well as a reduction in their subsequent number of females. Behavior and infection assays demonstrated that application of synthetic peptides Hg-FLP-22b (TPQGKWMRFa) and Hg-FLP-22c (KMAIEGGKWVRFa) significantly increased the head movement frequency and host invasion abilities in H. glycines but not in Meloidogyne incognita. In addition, the number of H. glycines females on the host roots was found to be significantly higher in Hg-FLP-22b treated nematodes than the ddH2O-treated control J2s. These results presented in this study elucidated that Hg-flp-22 plays a role in regulating locomotion and infection of H. glycines. This suggests the potential of FLP signaling as putative control targets for H. glycines in soybean production.The FMRFamide-like peptides (FLPs) represent the largest family of nematode neuropeptides and are involved in multiple parasitic activities. The immunoreactivity to FMRFamide within the nervous system of  Glycine max L. (Merr)] is an important and frequently consumed crop and serves as a major source of protein and oil worldwide. However, its yield is strongly affected by various pests and pathogens, including plant-parasitic nematodes (PPN). The soybean cyst nematode, Heterodera glycines Ichinohe, a sedentary and endoparasitic nematode, is one of the most damaging pests attacking soybean (H. glycines has been reported to be around US$1 billion in the United States (Zea mays L.) and the use of resistant host cultivars in the field. However, the limited availability of genetic resources for resistance to H. glycines and the genetic variation in the field population of H. glycines restrict the application of this method  also displayed a significant reduction in attraction and penetration of M. incognita in tomato root in the Pluronic gel system , thereby impairing their infection ability , with a C-terminal Arg-Phe-NHpeptides . FLPs haematodes . Only a ncognita , Meloidominicola , G. pall pallida , and Heta avanae . RNAi-me ability . Additiolp genes . More reM. incognita and M. graminicola; only in vitro physiological experiments have tested the proteolysis of two synthetic peptides (FLP-6 and FLP-14) and their effects on the behavior of H. glycines J2s , at least 20 flp genes are present in H. glycines. However, little is known about the functional characteristics of flp genes in H. glycines. In the present study, Hg-FLP-22 encoding gene was identified and cloned from H. glycines J2s, and its expression pattern was analyzed by quantitative real time (qRT)-PCR in different development stages. The functional characterization of Hg-flp-22 was studied in behavior, infection, and development of H. glycines via RNAi and exogenous application of synthetic peptides.To date, studies of FLP functions in PPN have primarily focused on root-knot nematodes, ines J2s . ChromatJ2 stage . Early sglycines . These o reports  and our Heterodera glycines race 5 was cultured on a susceptible commercial soybean cultivar “DongSheng1” in a greenhouse at 22–28°C in the Northeast Institute of Geography and Agroecology, Harbin, China. Cysts were collected from the soil and soybean roots from the culture pots. Eggs were released by manually crushing cysts with a rubber stopper and then harvested by pouring the solution through a 25 μm pore sieve. Collected eggs were further isolated from debris through sucrose  centrifugation and thoroughly washed in distilled water. Eggs were kept in 3 mM ZnSO4 solution in an incubator at 28°C for hatching. Fresh J2s were collected at 3–4 days and then used immediately in each experiment. A pure culture of M. incognita was maintained on tomato cv. “Zhongshu-4” in the greenhouse under the same conditions as mentioned above. Nematode eggs and J2s were collected as previously described by Hg-flp-22, was identified from this study of transcriptome sequence data of H. glycines J2s. Specific primers (Hg-flp-22 cDNA containing the predicted full-length open reading frame (ORF) and partial 3' untranslated region (UTR) using Phanta Super-Fidelity DNA Polymerase . The cycling conditions consisted of an initial denaturation at 95°C for 3 min, followed by 35 cycles of 95°C for 10 s, 60°C for 15 s, and 72°C for 30 s, followed by a final extension at 72°C for 5 min. The amplified PCR product was purified and cloned into pLB fast cloning vector , and then transformed into Escherichia coli DH5α chemically competent cells . Recombinant plasmids were isolated from at least four positive clones and sequenced by TSINGKE biological Technology . Return sequences were analyzed by using BioEdit version 7.2.5.A putative full-length cDNA encoding FLP-22, designated  primers  were desHg-flp-22 and its homologs across the nematode species were aligned using online multiple sequence alignment tools with default settings.To perform sequence homology comparisons, Basic Local Alignment Search Tool (BLAST) searches were conducted in the nematode non-redundant and expressed sequence tag (EST) databases at the National Centre for Biotechnology Information (NCBI) BLAST server by using BLASTX and BLASTN. The predicted protein sequences of 2O to make a 10 mM stock solution and was then aliquoted and stored at −20°C. For nematode behavioral assay, J2s of H. glycines or M. incognita were incubated for 15 min in 10 μM, 100 μM, or 1 mM solution of peptides in a PCR tube. For infection assay, J2s were added to 400 μl of peptides solution and incubated for 16 h at room temperature in the darkness. All of the peptides stock stored at −20°C were used within 1 week.Peptides QPAGGVKWMRFa (Hg-FLP-22a), TPQGKWMRFa (Hg-FLP-22b), and KMAIEGGKWVRFa (Hg-FLP-22c) were synthesized by SangonBiotech  at 95% purity. Each synthesized peptide was dissolved to sterilized ddHHg-flp-22 (250 bp) were synthesized and purified with a MEGAscript® RNAi Kit  according to theinstructions of the manufacturer using the primers with the T7 promoter sequence appended to the 5' end (gfp (720 bp) was synthesized from a plasmid containing gfp fragment and used as a non-native negative control. dsRNA soaking of J2s was performed as previously described by gfp gene and soaking buffer (without dsRNA) were set as the control. After 24 h incubation, J2s were washed four times with sterile distilled water through centrifugation to remove the external dsRNA. Additionally, dsRNA uptake efficiency was assessed by using fluorescein isothiocyanate  as a tracer in dsRNA buffer, and then, nematodes were observed under a fluorescence microscope .Double stranded RNA (dsRNA) of e 5' end . dsRNA oApproximately 500 dsRNA-treated J2s were used for total RNA isolation and qRT-PCR analysis of the transcribed abundance of the target gene. The remaining J2s were used to assess behavior, infection, and development of nematode.For head movement assay of nematode, approximately 100 J2s were incubated in peptides solution for 15 min. Then, J2s were transferred to a cell counting chamber  with four rooms (10 μl loading volume), and head movements were observed for a randomly selected individual nematode under a stereomicroscope . Head movement frequencies of J2s were determined following a definition previously described by g for 10 min at 4°C. The supernatant was then collected, filter sterilized , and used for chemotaxis assay  were homogenized in 4 ml sterile distilled water, and homogenates were centrifuged at 12,000 is assay . Fresh ris assay . The disHeterodera glycines attraction assays were conducted in six-well tissue culture plates containing Pluronic F-127 gel , root systems were harvested and the penetration ability of the nematodes was determined under a stereoscope through acid fuchsin staining. The number of females on the roots was counted under a stereomicroscope using a counter at 21 dai.Soybean seeds were surface-sterilized in 1.5% sodium hypochlorite for 15 min, rinsed thoroughly with tap water, and germinated for 4 days in darkness at 26°C, after which they were transferred to pots containing sterilized sand and soil (2:1) and grown in a growth chamber with a photoperiod of 16 h light (26°C) and 8 h dark (20°C) cycle. After 8 days, soybean seedlings were inoculated with 200 M. incognita inoculation, a similar procedure was used but with a tomato host plant. About 200 J2s were applied on each 2-week-old tomato seedling (cv. Zhongshu-4) which was grown and maintained in a sterilized soil mixture (1:1 mixture of sand and soil) in a 10 cm × 9.5 cm tray, and then, the number of nematodes that had penetrated the host roots was counted at 1 dai. During the course of the experiment, plants were watered four times a week, with one of the watering consisting of Hoagland’s nutrient solution  were used for RNA extraction. The infected roots were harvested at 3 and 12 dai, and the parasitic J2s and mixed J3s/J4s were extracted according to the method as described by −∆∆Ct method was applied to calculate the related expression of Hg-flp-22 in different life stages and dsRNA treatments  in different life stages of eatments . Hg-GAPD results . All expp < 0.05) was used for overall pairwise comparisons. The error bars in the figures represent the SD of means, and the significance level was set at p < 0.05.Data were subjected to one-way ANOVA using SPSS version 17.0 software . Tukey’s HSD test (flp-22 gene of H. glycines was identified based on our transcriptome sequence data of H. glycines J2s. The cDNA fragment of Hg-flp-22 (479 bp) containing the predicted full-length ORF and partial 3' UTR was amplified using specific primers , sharing 72% sequence identity; indeed, the identity of two peptides, QPAGGVKWMRFa and TPQGKWMRFa, in H. glycines and G. rostochiensis were 100%  using the expression levels in eggs as a reference by qRT-PCR. Hg-flp-22 was expressed at the highest levels in pre-parasitic J2s and adult males compared with other life stages. Although its transcript levels declined during the parasitic J2 and J3/J4 stages, it also exhibited, respectively, approximately 2.7- and 2.6-fold higher transcripts than those of the eggs. The lowest expression levels of Hg-flp-22 were found in adult females in contrast to its upregulation in early parasitic-stages of H. glycines  when compared with those in the soaking buffer without dsRNA. Non-native control dsRNA-gfp treatment did not result in a statistically significant reduction in the target transcript of Hg-flp-22  led to approximately 27.3% increase in the mean head movement frequency compared with the ddH2O-treated nematodes. Significant stimulatory effects of FLP-22c on the head movement of H. glycines were found at both 10 μM and 1 mM peptide solutions. In contrast, FLP-22a had no significant effect on head movement frequency (p < 0.05) and FLP-22c-treated  J2s penetrated into soybean roots compared with the ddH2O-treated control nematodes  and non-neuronal cells (URA and uv1) and was mainly focused on the anterior region. Notably, published data indicated that exogenous FLP-22 peptide of C. elegans has markedly excitatory effect on the pharyngeal action , CNR, and other nerve structures  significantly increased the head movement frequency of  species , also hancognita . Once hadepleted . Therefother PPN , suggestHg-flp-22 reduced the number of females in only one independent assay, indicating that the efficacy of the RNAi experiment may be transient. Although Hg-FLP-22b treatment increased the number of females, it is difficult to interpret this information as indicative of Hg-flp-22 function in parasitic stages because of the transient efficacy and short durability of RNAi and synthetic peptides, which have been reported in several studies (Hg-flp-22 was not effective in expressing Hg-flp-22-RNAi constructs of soybean hairy roots (data not shown), and further functional studies of Hg-flp-22 during H. glycines parasitism are needed.Lower infection ability of the RNAi-treated J2s may lead to a lower proportion of nematodes at the mature female stage. We found that silencing of  studies . UnfortuC. elegan by using heterologous cellular systems and genetic experiments . These data indicate a positive regulation of bioactive FLP-22 peptides in H. glycines behaviors in terms of increased body movement, penetration, and development. This study also deepens the basic understanding of FLP signaling in the early parasitism of H. glycines, which is the basis for the exploitation of the FLP system as putative control targets.In conclusion, this is the first functional characterization of FLP encoding genes in The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/YH conceived and designed the experiments and wrote the manuscript. JY, FP, SW, and YW performed the experiments. JY, FP, and YH analyzed the data. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Apoptosis assays demonstrated that miRNA-148a enhanced apoptosis before and after irradiation. Reporter assays revealed that c-Met was the direct target gene of miRNA-148a. An in vivo study indicated that miRNA-148a enhanced the irradiation-induced suppression of xenograft tumor growth (p < 0.01). miRNA-148a may be a biomarker of pCR following NACRT and can promote apoptosis and inhibit proliferation in CRC cells by directly targeting c-Met in vitro and enhancing tumor response to irradiation in vivo.Patients with locally advanced rectal cancer (LARC) who achieve a pathological complete response (pCR) to neoadjuvant chemoradiotherapy (NACRT) have an excellent prognosis, but only approximately 30% of patients achieve pCR. Therefore, identifying predictors of pCR is imperative. We employed a microRNA (miRNA) microarray to compare the miRNA profiles of patients with LARC who achieved pCR  with those who did not . The validation set confirmed that miRNA-148a was overexpressed in the pCR group (n = 11) compared with the non-pCR group (n = 40). Cell proliferation and clonogenic assays revealed that miRNA-148a overexpression radio-sensitized cancer cells and inhibited cellular proliferation, before and after irradiation ( Colorectal cancer (CRC) is the leading cause of cancer mortality worldwide, and approximately 30% of CRC cases are rectal cancer . NeoadjuPTEN and p21 [KRAS signaling and promoted radiosensitivity in lung cancer cells [VEGF by downregulating the pERK/HIF-1α/VEGF pathway, which may inhibit angiogenesis in CRC [MicroRNAs (miRNAs), non-protein-coding RNAs, regulate the expression of their protein-coding genes by degrading mRNA or repressing translation. miRNAs contribute to several critical biological functions, including carcinogenesis, cell proliferation, and apoptosis ,9. They  and p21 . In one er cells . miRNA-1s in CRC . In summThe study protocol was approved by the Institutional Review Board of Kaohsiung Medical University Hospital (KMUHIRB-02-11-2011). All participants signed an informed consent form. From May 2012 to March 2015, 51 patients with LARC treated with NACRT and radical resection were enrolled, and pretreatment cancer tissues were collected during colonoscopic biopsy and used for miRNA analysis. NACRT consisted of 50 Gy of irradiation concurrently with 5-fluorouracil-based chemotherapy. Radical resection was performed 8–12 weeks after NACRT. A pCR was indicated by the absence of any viable cancer cells in the primary tumor and lymph nodes. Patients were dichotomized according to their pathological response into pCR and non-pCR groups. The design of the identification of the candidate miRNA is shown in An miRNA microarray  containing probes for 667 human miRNAs was used to evaluate and compare the differential expression of miRNAs in the pCR and non-pCR groups. The mammalian U6 small nuclear RNA was used as the internal control for the detected miRNAs. PCR was performed using an Applied Biosystems 7900HT Real-Time PCR System, with default thermal cycling conditions on the ABI 7900 Sequence Detection System version 2.4.−ΔΔCt value.Total RNA was extracted from harvested cells using MasterPure Complete DNA and RNA Purification Kit Bulk Reagents . For the synthesis of cDNAs specific to miR-148a, a TaqMan MicroRNA Reverse Transcription Kit  was used. To determine the gene expression levels, qPCR reactions were performed with a TaqMan Universal Master Mix II kit . U6 small nuclear RNA was used as an internal control for miRNA-148a. Relative expression levels were normalized to U6 expression levels to yield a 2www.targetscan.org (accessed on 1 March 2017)) was used to identify the potential target genes of miRNA-148a. Only conserved sequences located in conserved target genes were considered. We used the Gene Ontology (www.geneontology.org (accessed on 18 May 2017)) software to detect the function of the target genes of miRNA-148a.The TargetScan program . A 1-cm bolus was placed on the top of the culture dish, and cells were irradiated with 6-MV photon beams at 600 MU/min [Human CRC cell lines, HT29 and HCT116, were purchased from the American Type Culture Collection  and the Bioresource Collection and Research Center , respectively. All cells were cultured in DMEM  supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin (Gibco) at 37 °C in a 5% CO0 MU/min .The HT29 and HCT116 cells were seeded in 24-well plates and transfected with 400 ng of miRNA-148a expression vector (pCDH-miRNA-148a) or a negative scrambled pCDH vector by using Lipofectamine 2000 transfection reagent . To select stably transfected cells, we cultured the cells for 4 weeks in selection media supplemented with 10 µg/mL puromycin . miRNA expression was measured using a TaqMan miRNA reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay  to confirm stable plasmid transfection. The transfected cell lines were then employed in the subsequent experiments.4 cells/well in 96-well plates. Subsequently, cells were irradiated with a single dose of 0, 2, 4, 6, or 8 Gy. After 72 h, the culture medium was removed and replaced with 0.5 mg/mL MTT and allowed to stand for 1 h at 37 °C for the formation of purple formazan. The precipitated formazan was dissolved with 100 µL of DMSO, and absorbance was measured at 570 nm with a microplate reader .Cell viability was examined using an MTT - 2,5-diphenyltetrazolium bromide reduction) assay. In brief, stable transfected HT29 and HCT116 cells were seeded at a density of 5 × 103 cells/well and exposed to 2–8 Gy of irradiation on day 2. After 10 days of incubation, the colonies were fixed with methanol/acetic acid (3:1) and stained with 0.5% crystal violet in 50/50 methanol/water for 20 min at room temperature. Next, the staining solution was carefully removed from each well and rinsed with water. Finally, the number of cell colonies with a size ≥1 mm was counted using ImageJ software (Java 1.8.0_172).For the clonogenic formation assay, transfected cells were seeded in 6-well plates at a density of 6 × 105 stained cells in each sample were detected through flow cytometry .After synchronization with serum starvation for 24 h, cells were irradiated at a dose of 4 Gy. Following 4 days of incubation, floating and adherent cells were harvested for cell cycle and apoptosis analysis. For cell cycle analysis, cells were fixed with 75% ethanol at 4 °C overnight. After cells were washed twice with PBS, they were resuspended with PI/Triton X-100  and incubated in the dark for 30 min. To detect apoptosis, we stained the harvested cells with PE-labeled Annexin-V/7-AAD, according to the manufacturer’s protocol . The signals of 1 × 10c-Met, caspase-3, poly (ADP-ribose) polymerase (PARP), and GAPDH were quantified using Western blotting. After 72 h of irradiation, the whole-cell extract was isolated using RIPA buffer . In brief, equal amounts of protein were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were then incubated with Trident Universal Protein Blocking Reagents  for 30 min at room temperature. This was followed by incubation with primary antibodies at 4 °C overnight. Target proteins were probed with the following antibodies: anti-phospho-c-Met, -c-Met, -caspase-3, -PARP . Anti-GAPDH  was used as a loading control for the whole-cell lysates. Subsequently, the membranes were incubated with a 1:5000 dilution of an HRP-conjugated antibody for 1 h at room temperature. Protein bands were developed using an enhanced chemiluminescence detection reagent, and signals were captured using the ChemiDoc MP Imaging System . ImageJ software was used for protein quantification.Met 3′UTR sequence (5′-AGGCCACAAAAACACUGCACUGU-3′) (cat. no. CW306396) or mutant 3′-UTR sequence (5′-AGGCCACAAAAACACACGUGACU-3′) (cat. no. CW306397) was cloned into the pMirTarget vector to construct the wild-type (WT) c-Met 3′UTR plasmid or the mutant c-Met 3′UTR luciferase plasmid . Cells (1 × 105) were seeded into 24-well plates for 1 day and cultured until the cells reached 70–90% confluence. Subsequently, cells were transfected with WT or mutant-3′UTR luciferase plasmid (0.5 µg) using Lipofectamine 3000 reagent , according to the manufacturer’s instructions. Luciferase activity was measured 48 h after transfection using a dual-luciferase reporter assay kit. Firefly luciferase activity was normalized to Renilla luciferase activity.The predicted miRNA-148a binding site of the 6 cells were subcutaneously injected. On days 12, 15, and 17 after the injection, tumors were irradiated with 15 Gy in three fractions. The tumor size (mm3) was measured three times a week and calculated as (length × width2)/2. Mice were killed 30 days after the injection of tumor cells.For tumor implantation, 6-week-old male Balb/c nude mice were obtained from BioLasco Taiwan . All animal experiments adhered to the protocols of the Institutional Animal Care and Use Committee of Kaohsiung Medical University  and were performed according to the Guiding Principles for the Care and Use of Laboratory Animals. The mice were acclimatized for 1 week after arrival under a 12 h:12 h dark/light cycle at 22 ± 1 °C with ad libitum access to food and water. The cells were harvested by trypsinization and washed twice with ice-cold serum-free medium, followed by resuspension in 100 μL of serum-free medium. Into the right flank of each mouse, 2 × 10t tests were conducted to analyze the differences in the expression levels of miRNAs in the pCR and non-pCR groups. Kaplan–Meier survival curves were plotted, and a log-rank test was performed to compare time-to-event distributions. Overall survival (OS) was calculated from the date of diagnosis to death from any cause, and disease-free survival (DFS) was calculated from the date of diagnosis to any recurrence. Receiver operating characteristic (ROC) curve analysis was employed to identify the cutoff value of miRNA-148a to predict pCR. All analyses were performed using JMP software . A p of <0.05 was considered significant.All values are presented as means ± standard errors of the mean of at least three independent experiments. Student’s The patients’ clinicopathologic characteristics are presented in p = 0.042) and miRNA-148a (p = 0.025) displayed a more significant overexpression in the pCR group compared with the non-pCR group. Therefore, we selected miRNA-148a as a predictor of pCR and subsequently examined the biological functions of miRNA-148a through in vitro and in vivo studies.To identify the miRNAs associated with a pCR to NACR, 10 cancer tissues obtained from patients with LARC before NACRT were collected for miRNA microarray analysis. Through this analysis, changes in miRNA expression profiles between the pCR group (n = 5) and the non-pCR group (n = 5) were measured. We observed that 22 miRNAs were differentially expressed in the tissues of patients in the pCR and non-PCR groups. Specifically, 14 were upregulated in the pCR group and 6 were downregulated in the pCR group . Of the p < 0.05, p < 0.05). Due to the upregulation of miRNA-148a identified in the pCR group and the inhibition of cell growth following the overexpression and irradiation of the transfected miRNA-148a, we postulated that miRNA-148a could modulate radiosensitivity and, thus, increase the likelihood of a pCR. To further confirm the role of miRNA148a in the enhancement of CRC cells’ radio-sensitivity, we performed clonogenic assays, which revealed that miRNA-148a overexpression significantly reduced colony formation ability following irradiation, compared with that in cells transfected with scrambled miRNAs . By contrast, miRNA148a overexpression corresponded to a substantial reduction in the proportion of cells in the G1 phase, whereas miRNA148a overexpression exerted no influence on S-phase alterations.To determine the effects of miRNA-148a on cell cycle alterations, flow cytometry was conducted. A significantly increased G2/M arrest was observed in cells transfected with the miRNA148a mimic after 24 h; furthermore, the increase in the proportion of cells in the G2/M phase was more prominent in cells overexpressing miRNA148a and subjected to 4-Gy radiation (p < 0.05). The percentage of apoptotic cells in the miRNA148a overexpression group after radiation was significantly higher than that in the control group . Cleaved PARP-1 is a well-established apoptotic marker and indicates an apoptotic-specific event [p < 0.01). Taken together, these findings indicate that miRNA-148a can enhance radiation-induced apoptosis.To explore the effects of miRNA-148a on apoptosis, HT29 cells with miRNA148a overexpression were exposed to 4 Gy of radiation and subjected to Annexin-V/7-AAD staining for of the evaluation of apoptosis. miRNA-148a overexpression had a 37% higher increase in apoptotic cells compared with the negative control (NC) groups  mice injected with cells containing pCDH-NC; (2) mice injected with cells containing miRNA-148a; (3) mice injected with cells containing pCDH-NC and receiving tumor irradiation; and (4) mice injected with cells containing miRNA-148a and receiving tumor irradiation  for pCR, and an miRNA-148a expression level of 1.8 corresponded to an optimal sensitivity of 55% and specificity of 58%. We then determined whether the cutoff value was associated with patient outcomes, observing that survival was significantly reduced in patients with low miRNA-148a expression. The 5-year OS rates in the high miRNA148a expression and low miRNA148a expression groups were 95.5% and 76.7%, respectively (p = 0.0463; p = 0.0018; To examine the clinical significance of miRNA-148a expression in predicting a pCR in patients with LARC, we performed ROC analysis to identify the miRNA-148a cutoff value. The area under the curve was 0.65 (c-Met. Taken together, the results indicate that miRNA-148a can serve as a potential predictive biomarker to guide the watch-and-wait strategy suggested for patients with LARC following NACRT.Studies have indicated that NACRT improves prognosis in patients with LARC; however, treatment response varies from total regression to total resistance ,3. As pamiRNAs play an integral role in cancer development and progression and can be classified as oncomiRNAs or tumor suppressor miRNAs on the basis of their biological functions . MoreoveOwing to the overexpression of miRNA-148a in the pCR group compared with that in the non-pCR group, this was regarded as associated with pCR. miRNA-148a, which is located at chromosome 7p15, functions as a tumor suppressor miRNA and is involved in various cancer-related processes, including cell proliferation, invasion, migration, and apoptosis . Studiesc-Met in the HCT116 cells, but not in the HT29 cells. This may be attributable to the differences in their mutational statuses. Bacco et al. demonstrated that the irradiation-induced expression of c-Met was related to the activation of ATM and NF-kB [NF-κB activation and KRAS mutation [KRAS is a mutation in HCT116 cells but is WT in HT29 cells [c-Met upregulation was prominent in the HCT116 cells and not the HT29 cells because NF-κB activation might be related to KRAS mutation.To investigate whether miRNA-148a functioned consistently in cells bearing distinct gene mutations, we examined the biological functions of miRNA-148a by using two CRC cell lines with distinct mutational statuses . HT29 cend NF-kB . Lin et mutation . KRAS is29 cells ; therefoThe role of miRNA-148a in the regulation of radiosensitivity has rarely been investigated. Wang et al. found that SNHG12, a class of long noncoding RNAs, mediated the radiosensitivity of cervical cancer cells through the miRNA-148a/CDK1 pathway . Lopez-Bc-Met is common in gastrointestinal cancer and is considered to be associated with tumor progression and poor survival. c-Met is a receptor tyrosine kinase that binds to hepatocyte growth factor and triggers various cancer-associated processes, including proliferation, angiogenesis, invasion, and epithelial–mesenchymal transition [c-Met overexpression in patients with CRC has been associated with reduced survival and increased risk of distant metastasis [c-Met is an miRNA-148a target gene in CRC cells. Furthermore, the combination of miRNA-148a overexpression and irradiation significantly inhibited the expression of c-Met, which subsequently promoted apoptosis. c-Met is associated with radio-resistance. In one study, its inhibition led to radio-sensitization in various cancers, including CRC [c-Met pathway sensitized glioblastoma to irradiation, both in vitro and in vivo [c-Met inhibitor, radio-sensitized KRAS-mutant CRC cell lines, suggesting that crizotinib can be prescribed to patients with CRC requiring radiotherapy [c-Met overexpression increased invasiveness and inhibited apoptosis in breast cancer cells and that c-Met inhibitors reversed these effects, indicating radio-sensitization in cancer cells by inhibition of c-Met [c-Met overexpression in surgical specimens resulted in poor relapse-free survival [c-Met by miRNA-148a enhanced radiosensitivity in tumor cells. Taken together, these results suggest that miRNA-148a, which downregulates c-Met expression, is a potential therapeutic agent and radiosensitizer in patients with LARC receiving NACRT. Future studies should confirm the role of miRNA-148a in this regard and address the relevant clinical implications.Aberrantly regulated ansition . c-Met otastasis . The preding CRC . Lal et  in vivo . Cuneo eotherapy . Bacco eof c-Met . Kawamursurvival . Consistc-Met signaling pathway of mediating radiosensitivity was not completely explored in this study. Activation of c-Met induces various cellular signaling pathways and consequent biologic functions. A better understanding of the c-Met signaling pathway would help the development of new therapeutic agents. Therefore, the detailed mechanisms of c-Met-mediated cellular response to irradiation warrant further studies. Despite these limitations, we consider that miRNA-148a is a potential predictive biomarker and may play an important role in personalized therapy for patients with LARC.Some limitations of this study need to be addressed. First, the number of patients was relatively small. A larger cohort is essential to validate the predictive value of miRNA-148a in LARC. Second, the detailed c-Met in vitro and enhancing tumor response to irradiation in vivo. Further studies on the clinical implications and regulatory mechanism of miRNA-148a are warranted to determine its role in LARC treatment.In this study, we demonstrated that miRNA-148a is a potential biomarker for predicting pCR following NACRT and that it was associated with favorable oncological outcomes in patients with LARC. miRNA-148a overexpression promoted apoptosis and inhibited proliferation in CRC cells by directly targeting


Escherichia coli and Staphylococcus aureus viability in 2 h can be produced by simple measures using a commercial acrylic topcoat for wood surfaces. We characterize the surfaces taking into account cyclic wear and variable environmental conditions. The light-induced antibacterial and photocatalytic activities of the surfaces are enhanced by short-term cyclic wear, indicating their potential for prolonged effectivity in long-term use. As the produced surfaces are generally more effective at higher relative air humidity and silver-containing surfaces lost their contact-killing properties in dry conditions, it is important to critically evaluate the end-use conditions of materials and surfaces to be tested and select application-appropriate methods for their efficacy assessment.In the context of healthcare-acquired infections, microbial cross-contamination and the spread of antibiotic resistance, additional passive measures to prevent pathogen carryover are urgently needed. Antimicrobial high-touch surfaces that kill microbes on contact or prevent their adhesion could be considered to mitigate the spread. Here, we demonstrate that photocatalytic nano-ZnO- and nano-ZnO/Ag-based antibacterial surfaces with efficacy of at least a 2.7-log reduction in  Bacterial infections are exerting a significant impact on public health. According to Global Health Metrics , microbi2 surfaces may, in addition to bacterial killing, also lead to the photooxidation of bacterial debris and, thus, the removal of dead bacterial mass from such surfaces, leading to the possibility for their extended use [The most effective way to prevent the spread of microbial pathogens is to stop their transmission from person to person or via cross-contaminated surfaces. The latter is of significant importance as even up to 40% of microbial infections in hospital settings may spread through fomites, or microbe-contaminated materials ,6. Therended use . Besidesnded use ,17, we dnded use , the lig2+ and Ag+ to the antibacterial action of our surfaces. Altogether, our results are expected to pave the way towards the use of nano-ZnO- and nano-ZnO/Ag-based surface coatings in fit-for-purpose antimicrobial surface applications.We have already demonstrated the antimicrobial efficacy of nano-ZnO/Ag composite materials both under UVA illumination and in dark conditions. Our previous studies have demonstrated 99.9% antibacterial activity of the surfaces in laboratory conditions under UVA that corresponded spectrally and in terms of intensity to natural solar light . HoweverNano-ZnO and nano-ZnO/Ag particles were prepared similarly to our previous works ,17. Brieα, zinc Kα and oxygen Kα were extracted to visualize their elemental distribution.Scanning transmission electron microscopy–energy-dispersive X-ray spectroscopy (STEM–EDX) analysis was used to characterize the morphology and elemental content of nano-ZnO/Ag. First, 200 μg/mL particle suspension was deposited onto a 400-mesh holey carbon-coated copper grid  and analyzed using a Titan Themis 200 Cs corrected transmission electron microscope , Waltham, MA, USA) at 200 kV. EDX signal of the particles was collected with the SuperX silicon drift detector  in STEM mode. The high-angle annular dark-field (HAADF) STEM image was combined with EDX element mapping and signals for silver L3.The concentration of Zn and Ag in nano-ZnO and nano-ZnO/Ag suspensions used for preparing nanoparticle-covered substrates and embedded nanoparticle-based coatings was analyzed using total reflection X-ray fluorescence  for Zn and an atom absorption spectrometer  for Ag, followed by acidification of nanoparticle suspensions with 1% HNOFirst, 25 × 25 mm squares of plywood  were used as substrates to be covered with acrylic-embedded nano-ZnO and nano-ZnO/Ag. Acrylic clear matt commercial two-component topcoat meant for wooden surfaces  was used as the acrylic matrix to embed the prepared nano-ZnO and nano-ZnO/Ag particles. To mix nano-ZnO or nano-ZnO/Ag to TZ9310/00, the particles were first dispersed in the solvent  used in the topcoat mixture and then mixed with the rest of the topcoat components. Prior to covering the wooden surfaces with nanomaterial-supplemented acrylic matrix, the surfaces were undercoated  and left to dry in ambient laboratory conditions for a week. Nano-ZnO and nano-ZnO/Ag supplemented topcoat was then evenly applied onto the surfaces using a brush. Brushing was used to mimic a possible real-life approach for applying the coating. Hence, the thickness of the coating was a result of the chosen coating technique and was not further optimized. The surfaces were left to dry at ambient laboratory conditions for a week before subsequent analysis. Wooden surfaces covered with undercoat and topcoat without any nanomaterial addition were used as negative controls.3 for 1 h, after which the solution was diluted with water and analyzed using TXRF or AAS. Bare cover glasses were used as controls in these experiments.For “nanomaterial controls” in antimicrobial and photocatalytic experiments, glass surfaces with (matrix-free) nano-ZnO and nano-ZnO/Ag particles were used. For this, nano-ZnO and nano-ZnO/Ag suspensions were spin-coated onto 25 × 25 mm square cover glasses . The surfaces were subsequently heated at 200 °C for 6 h (similarly as described in our former study ) to imprFor scanning electron microscopy (SEM) analysis, the upper layer of matrix-embedded nano-ZnO- and nano-ZnO/Ag-covered plywood surfaces was removed and attached onto a 45-degree angled SEM pin stub using conductive silver paint. Top view and cross-section of the initial samples and samples after 5 and 10 reuse cycles were imaged by Nova NanoSEM 450 (Thermo Fisher Scientific Inc. (formerly FEI), Waltham, MA, USA) at 15 kV using a low vacuum detector at 100 Pa.−8 mbar, and then heated twice to 60 °C for 1 hour. XPS measurements were conducted by using an electron energy analyzer, the Scienta SES-100, and a non-monochromatized twin-anode X-ray source, the Thermo XR3E2 (Mg Kα 1253.6 eV and Al Kα 1486.6 eV). The Ar+ ion sputtering was performed in ultra-high vacuum conditions at 10−8 mbar. The duration of the Ar+ beam exposure was 15 min and the Ar+ current was 10 mA. The spot size was adjusted to cover the sample. The binding energy scales for the XPS experiments were referenced to the binding energy of C 1s (284.8 eV) photoemission line. Data analysis was carried out by using CasaXPS software  [Chemical state and electronic structure of the matrix-embedded nano-ZnO and nano-Ag/ZnO on wood surfaces were investigated with X-ray photoelectron spectroscopy (XPS). Samples were first degassed in vacuum, until the base pressure was 1 × 10uth, UK) . Zn 2p, Wavelength-dispersive X-ray fluorescence (WD-XRF) spectroscopy was used to determine the Zn concentration in matrix-embedded nano-ZnO- and nano-ZnO/Ag-covered plywood surfaces. The WD-XRF 4 kW instrument  was calibrated using LabKings SM68 Standard 1 as a reference. The set amount of standard was pipetted onto control coatings (pure topcoats without added nanoparticles) and the solvent was evaporated by heating at 70 °C in an exicator. Calibration curve was established using 5 reference concentrations.Release of Zn and Ag from matrix-embedded nano-ZnO- and nano-ZnO/Ag-covered surfaces was measured in conditions comparable to antimicrobial tests. First, 20 μL of test medium was pipetted onto the surfaces and incubated under 20 mm × 20 mm × 0.05 mm polyethylene (PE) film for a specified duration. The liquid was then washed off with 2 mL deionized water. Zn content in the wash-off was measured by TXRF  and Ag content by AAS .The sessile drop method  in combiEscherichia coli DSM1576 (ATCC8739) was ordered from the German Collection of Microorganisms and Cell Cultures  and Staphylococcus aureus ATCC6538 was acquired from the National Institute of Chemical Physics and Biophysics . For bacterial suspensions used in the tests described below, bacteria were cultivated overnight on nutrient agar plates , collected by using a sterile inoculation loop and suspended in exposure medium  followed by adjusting cell density by means of optical density measurement and further dilution in exposure medium based on prior calibration. Bacteria were harvested from the surfaces by submerging the surfaces in 15 mL of toxicity-neutralizing soybean casein digest broth with lecithin and polyoxyethylene sorbitan monooleate  in 50 mL centrifugation tubes, followed by vortexing the tubes for 30 s at full power, serial dilutions in phosphate-buffered saline , drop-plating on NA plates and incubation at 30 °C. Colonies of E. coli were counted 24 h and S. aureus 48 h after plating. Results are expressed as log-transformed CFU counts. All aqueous media were prepared and/or diluted in sterile deionized water and autoclaved to sterilize. Bovine serum albumin  was filter-sterilized and added to soil load solution after autoclaving and cooling to room temperature. Gram-positive and Gram-negative model organisms routinely used in standard protocols were selected for the assessment of antibacterial efficacy. E. coli and 0.070 for S. aureus, resulting in approximately 3.5 × 105 CFU/cm2. Then, 20 μL of the prepared inoculum suspension was applied to 25 × 25 mm sample surfaces, covered by 20 × 20 × 0.05 mm PE film and incubated in the dark or exposed to UVA in parallel. PE cover film was used to attain even thin coverage of bacterial suspension with good contact between microbes and the surface and even UVA exposure, as well as to ensure similar humidity conditions on the predefined test area. Exposures were carried out in a humid environment on a bent glass U-rod over sterile wet filter paper covered by a Petri dish in dark conditions or 1.1 mm thick UVA-transmissive borosilicate moisture preservation glass under UVA illumination. Temperature (22 °C) and lighting  were achieved in a climate chamber, the MMM Climacell 111 EVO, equipped with a UVA/Vis Combi light tray . Exposures were terminated, cells harvested and viable counts determined as described above.Antibacterial testing was carried out using a test protocol modified from ISO 22196 and ISO 27447 standard procedures adapted to the studied materials and the need for higher throughput due to several materials and time points studied, as described by Visnapuu et al. . Precult2 at 315–400 nm spectral range achieved in climate chamber MMM Climacell 111 EVO as specified above). Exposures were terminated, cells harvested and viable counts determined as described above.Bacterial suspensions were prepared as described above, but applied to the surfaces in 1 μL droplets in the same volume and final cell counts per surface. The surfaces were incubated under the same lighting conditions as above but left uncovered and kept at different relative air humidities. Only 1 h time point was collected at each humidity condition, temperature (22 °C) and lighting  of ZnSO−5 M MB aqueous solution was pipetted onto the prepared surfaces and exposed to UVA irradiation or kept in dark conditions for control in a climate chamber . The pH of the initial MB solution was 5.3, which is close to the recommended pH value (pH 5.5) for the solution [max = 355 nm) was used and the light intensity at test surface height was 2.8–3.2 W/m2 at 315–400 nm spectral range (measured using Delta Ohm UVA probe).The degradation of methylene blue (MB) as a model for an organic contaminant was used to determine the level of photocatalytic activity. First, 2 mL of 1.6 × 10solution . After pp < 0.05) differences and log reductions are mentioned in the text, unless stated otherwise.One-way or two-way ANOVA analysis followed by Tukey post-hoc test was used to detect significant differences in multiple comparisons at 0.05 significance level using GraphPad Prism 9.1.2 . Log-transformed data at or above the detection limit of 3 colonies counted in undiluted plated drop were used for the analysis of CFU counts. Only statistically significant (α) in blue, zinc  in violet and oxygen  in red. The HAADF-STEM images of ZnO nanoparticles (not shown on figure) are similar, but without small Ag-based nanoparticles. The homogeneous distribution of elemental Zn and O over the rod-shaped nanoparticles confirms that these nanoparticles are based on ZnO. The distribution of Ag demonstrates that silver nanoparticles are much smaller than ZnO nanorods and are located on the surfaces of larger ZnO nanoparticles, as expected. The synthesized nanoparticles of ZnO/Ag composites are shown in Elemental analysis indicated that, in the suspensions of nano-ZnO and nano-ZnO/Ag that were used to prepare the nano-ZnO and nano-ZnO/Ag acrylic matrices, the Zn concentration was 50,000 µg/mL (6.2 wt% ZnO), and in the nano-ZnO/Ag suspension, the Ag concentration was 350 µg/mL (0.4 mol% ZnO).2) determined using XRF. The Zn content in both nanoparticle-based coatings was similar and much higher compared to the control (pure matrix-covered substrate). It is important to note that XRF provides information about the elemental content of bulk material, i.e., in the case of our samples, XRF collected information from the whole coating thickness, along with some signals from the plywood substrate.2+ oxidation state, as expected in the case of ZnO. More detailed XPS spectra for the same samples are shown in + sputtering, i.e., “as prepared” and after the removal of the topmost layer of sample with Ar+ ion bombardment. No Ag signal was detected in either case, likely due to the insufficient sensitivity of XPS to the low levels of Ag present in the ZnO/Ag composites.XPS was used to characterize the surface regions of the samples. Differently from XRF, X-ray photoelectron microscopy is a surface-sensitive method and it collects data only from the topmost parts (some nm) of the surface. Since microbe/surface interaction and also dissolution processes take place at the surface region, XPS data are very informative in this context. From the overview spectra of nano-ZnO- and nano-ZnO/Ag-containing coatings on plywood substrates a, the prIn order to assess the antibacterial activity of the matrix-embedded nano-enabled surfaces, antibacterial testing was performed following an in-house modification of ISO 22196 and ISO 27447 standard methods for higher throughput due to several materials and time points tested. The mentioned methods are based on a thin layer of liquid bacterial inoculum in contact with a large surface area  and are p < 0.0001) and >2.7 logs (p < 0.0001) of reduction in the viability of E. coli and S. aureus, respectively  and 1.1 (p = 0.0002) log reductions in viability, respectively  and 1.2 (p = 0.0009) log reductions on nano-ZnO/Ag compared to the control at 1 and 4 h, respectively, while the corresponding reduction values for S. aureus were non-significant, at 0.1 logs (p = 0.9985) after 1 h and 1.1 logs (p = 0.0002) after 4 h of dark exposure. No statistically significant antibacterial activity of matrix-embedded nano-ZnO towards S. aureus after 4 h dark exposure was detected and its activity towards E. coli was biologically negligible .The antibacterial activity of the matrix-embedded nano-ZnO/Ag surfaces was similar, with a statistically significant but biologically moderate effect towards both ectively a,b. HoweE. coli and S. aureus viability  than matrix-embedded nano-ZnO/Ag. Respective statistically significant differences between these two bacterial species on nano-ZnO-enabled surfaces could not be determined. There were no statistically significant differences between the matrix-embedded NP surfaces and bare NP surfaces in dark conditions. As stated above, we have previously tested the antimicrobial activity of solid surfaces based on nano-ZnO and nano-ZnO/Ag heat-annealed to glass carriers in different coverage densities ,17 and s2+ release under UVA illumination. Mean concentrations of Zn in the test system did not reach MBC values, suggesting that any toxic effects of the surfaces in dark conditions could not be caused by ionic toxicity but rather surface contact or the combined toxic effects of the multimodal surfaces. Based on the MBC values, E. coli was clearly more sensitive to ROS toxicity caused by hydrogen peroxide  in E. coli viability compared to the control was registered in ISO conditions. Due to the fact that the mean Ag concentration in the system for the modified ISO test system was below the bactericidal values (Staphylococcus aureus [Enterococcus faecium [S. aureus (E. coli (1–2 logs decrease in viability) does not seem to be affected by RH (not quantifiable at 15% RH). Towards S. aureus, the photo-induced antibacterial activity of nano-ZnO does not seem to follow a linear change and demonstrates the highest activity at the lowest RH and in wet ISO conditions but not at moderate RH, with 1.7 (p < 0.0001) and 1.9 (p < 0.0001) log reductions in viability, respectively. Apart from the effect of active agents in the surfaces, it is evident that low-intensity UVA exposure is more phototoxic to both bacteria in dry conditions , with respective antibacterial activities of 1.5 logs (p < 0.0001) reducing to a non-significant 0.5 logs. However, the antibacterial activity of the nano-enabled surfaces in UVA-irradiated conditions increased after 10 reuse cycles, with at least a 1.0 log reduction in bacterial viability for the nano-ZnO  and a moderate 0.9 log difference for nano-ZnO/Ag . While organic dye degradation measurements showed variable results after five reuse cycles, a statistically significant increase in photocatalytic activity was detected for both nano-ZnO  and nano-ZnO/Ag surfaces . The nano-ZnO/Ag surfaces were more active than the nano-ZnO surfaces in the case of the virgin surfaces  and after 5  and 10  reuse cycles. The small decrease (less than 3% in most cases) in MB UV–Vis absorbance intensity in the case of pure matrix-covered wooden surfaces can be associated with MB adsorbance onto the coating and not with dye degradation  and, thus, they were able to exhibit antibacterial effects. However, the measured antibacterial effect was significantly slower than that of bare nanoparticles. The surfaces based on matrix-embedded nano-ZnO and nano-ZnO/Ag demonstrated substantial antibacterial activity under UVA illumination in standard wet conditions, with at least a 2.7-log reduction in bacterial viability in 2 h. The addition of Ag to the surface coating matrix granted moderate antibacterial activity also in dark conditions after 4 h. Ag also enhanced photocatalysis and Zn release from the matrix-embedded surfaces under UVA illumination but did not affect the antibacterial properties under UVA. Neither metal release from the surfaces nor photocatalytic activity could explain the antibacterial effect in all conditions, confirming the multimodal antibacterial action of the surfaces resulting from photocatalysis, Zn and Ag ion release and contact killing. Antimicrobial efficacy testing at variable application-relevant relative air humidity revealed that better antibacterial efficacy was achieved at higher humidity and silver lost its contact-killing properties in dry conditions, highlighting the need to use a methodology that best reflects the end-use conditions. The UVA-induced antibacterial and photocatalytic activities of the surfaces were enhanced by short-term cyclic wear, indicating the potential for prolonged effectivity in real use situations.As a future outlook, antimicrobial surfaces with sufficient efficacy have certainly huge potential in maintaining good hygiene and a healthy environment, especially in vulnerable settings and public spaces. However, such surfaces should be developed in a sustainable manner, considering the potential environmental effects, including the long-term durability of the surfaces, as well as the minimization of the risk of the development of antimicrobial resistance.


Feline coronavirus (FCoV) is endemic in cat populations worldwide. Persistently, subclinically infected cats play a significant role in spreading the infection. Testing fecal samples of cats may facilitate efforts to decrease the viral burden within a population. Real-time RT-PCR is highly sensitive and specific for the detection of FCoV but must be performed in a fully equipped laboratory. A simple and accurate assay is needed to identify FCoV at the point-of-need. The aim of this study was to develop a rapid FCoV detection assay based on isothermal amplification technology, i.e., reverse transcription-recombinase polymerase amplification (RT-RPA). Primers were designed to target the highly conserved 3′ untranslated region of the 7b gene. Running on a constant temperature of 42 °C, reverse transcription as well as DNA amplification and detection was achieved in a maximum of 15 min. A probit analysis revealed a detection limit of 58.5 RNA copies/reaction. For cross-detection, nucleic acids from 19 viruses were tested. Both RT-RPA and real-time RT-PCR showed cross-detection with canine coronavirus and transmissible gastroenteritis virus, but not with other pathogens. To evaluate clinical performance, RNA was extracted from 39 fecal samples from cats. All samples were tested simultaneously with real-time RT-PCR resulting in a RT-RPA sensitivity and specificity of 90.9% and 100%, respectively. RT-RPA can be considered a promising simple method for rapid detection of FCoV. Alphacoronavirus, family Coronaviridae, and order Nidovirales [Feline coronavirus (FCoV) is an enveloped single-stranded positive-sense RNA virus, belonging to the genus ovirales . Based oovirales ,3. The covirales . Since tovirales . FCoVs aovirales . Serotypovirales ,8,9,10. ovirales . It is kovirales ,13,14. Oovirales ,16,17. Iovirales . Furtherovirales ,14,17,19ovirales  lead or ovirales . The cliovirales . Cats wiovirales . New stuovirales ,24.Several molecular assays based on RT-PCR, either conventional or real-time, were established for the detection of FCoV ,26,27,28In this study, the RT-RPA assay for rapid FCoV detection was developed based on the highly conserved 3′UTR of the 7b gene. The sensitivity and specificity to detect FCoV as well as cross-detection was determined. The assay’s clinical performance was validated using fecal samples. All results were compared with real-time PCR as a reference method.5 and 102 of the RNA molecular standard/μL  and Reverse Primer 3 (RP3)  . FP1 + R 3 (RP3)  and were3 to 100 copies/reaction)  (The RT-RPA assay’s limit of detection was determined using various concentrations of an in vitro-transcribed RNA molecular standard (5 × 10eaction) a. Out of(95% CI) .6 to 22.5 × 100 copies/reaction of RNA was revealed. The FCoV RT-RPA assay amplified down to 22.5 × 100 copies/reaction of the genomic RNA , transmissible gastroenteritis virus strain 70 RVB) .The clinical performance of the RT-RPA assay was examined using 39 fecal samples . The RT-The 3′UTR end of three samples, including one false negative and two positive identified samples, were sequenced. The obtained sequences were compared using Geneious Prime to evaluate whether changes in the nucleotides of one sample were the cause of false negative results in the RT-RPA. The alignment of the sequences showed no differences between the three samples in the primer and probe region.FCoV infection poses a high threat to cats, especially kittens, due to the possible development of FIP. To prevent further spread of the infection, the development of a fast and sensitive surveillance system for FCoV shedding is a necessity. In this study, an isothermal RT-RPA assay for rapid detection of FCoV within 15 min was developed and its diagnostic utility was compared to real-time RT-PCR. A limit of detection of 58.5 copies/reaction and a clinical sensitivity and specificity of 90.9 and 100%, respectively, were achieved. Of all viruses, RNA viruses in particular have a high tendency to mutate . To deteAs no strict rules for RPA primer design are prescribed, several primer combinations were tested in the present study. Many recommendations can be followed from the kit’s producer  and/or pCoronaviridae or were common pathogens in cat and dog populations. Both the new assay and the real-time RT-PCR-amplified strains of FCoV, as well as one strain of canine coronavirus (CCoV) and one strain of transmissible gastroenteritis virus (TGEV), are all alphacoronaviruses. Antigenic cross-detection is limited to species that belong to the same genus [The cross-detection to other pathogens was determined by using 19 different viruses, which either belonged to the family me genus ,46,47. Tme genus . In thisme genus .Of all fecal samples detected positive in real-time RT-PCR (n = 22), twenty samples with Ct values between 15 and 36 in real-time RT-PCR were correctly identified as positive in the RT-RPA assay. The other two positive samples in real-time RT-PCR were false negative in the RT-RPA assay. Generally, false negative results can be due to low viral load, as well as mutations at the targeted sequence or inhibitory effects. In this case, the latter is unlikely to be the reason, as RPA is known for its robustness to inhibitory effects . Low virAnother very popular isothermal amplification assay is the loop-mediated isothermal amplification (LAMP). LAMP is based on the use of a DNA polymerase with strand displacement activity, making amplification possible at a constant temperature (60–65 °C) for 30–60 min . LAMP reImmunochromatography tests for the detection of FCoV antigen are commercially available from various companies. The sensitivity and specificity in comparison to RT-PCR is around 95% ,54. UnfoThe standard molecular diagnostic methods for RNA virus detection remains real-time RT-PCR. This method, however, is time-consuming and requires a well-equipped laboratory, rendering it impractical as a routine point-of-need test. To overcome these drawbacks, rapid PCR systems have been developed recently ,56,57. TOne RPA assay for detection of FCoV was developed based on the membrane gene as the target region , which wTo conclude, the FCoV-RT-RPA was proven a rapid and sensitive assay for the detection of FCoV in the extracted samples. The deployment of rapid point-of-need tests and following measures would lead to a significant reduction of FCoV in cat populations. The easy handling of the RT-RPA assay makes repeated testing possible for identifying intermittent shedders.In total, 39 archived samples were used. Three samples were used from a pool of archived samples from cats with an unknown infection status collected during a surveillance study in Leipzig approved by the Landesdirektion Sachsen: A 19/17. A total of 36 fecal samples were collected during routine diagnosis. The owners had approved the use of the leftovers from the samples for research purposes. To test for cross-detection, inactivated viruses which were not available in our laboratory were provided by Friedrich Loeffler Institute, Robert Koch Institute and Charité Berlin, Germany. Viral RNA from all samples was extracted using the viral RNA Mini Kit  according to the instructions of the manufacturer.7–100 RNA molecules/μL were prepared.RNA standard  was used to determine the assay’s analytical sensitivity. First, a DNA strand was produced by Thermo Fisher Scientific GENEART  with the T7 promotor attached at the 5′-end (5′–TAATACGACTCACTATAG–3′). The DNA was transcribed into RNA using HiScribe T7 Quick High Yield RNA Synthesis Kit  following the manufacturer’s instructions. DNase treatment was applied to remove the background DNA using DNase I (2000 U/mL) . RNA quantification was performed by Qubit RNA BR Assay Kit from Thermo Fisher Scientific . Ten-fold serial dilutions ranging from 10The real-time RT-PCR assay was performed using a published protocol  on the SThree forward and three reverse primers as well as Exo-probe  were pro3–100 RNA copies per μL) as well as various concentrations of viral whole-genome extracted from cell culture supernatant (1.7 × 106 to 4.5 ×100 RNA copies per μL). Per reaction, 5 μL of the RNA was used. For determining the analytical sensitivity, the viral RNA dilution series was tested with and without host background nucleic acid extracted from real-time RT-PCR FCoV negative samples . Using RStudio version 1.3.1093  [The analytical sensitivity of the RT-RPA assay was determined using serial dilution of the molecular RNA standard (10 States) , a probi States) ). To detThe sequencing of the 7b gene of three samples was conducted based on a previous publication of Lin et al.  RT-PCR wFor electrophoresis, 8 μL of each sample was analyzed using a 1.5% agarose gel. The amplicon was purified  and then sequenced by Eurofins Genomics . Geneious prime  was applied for data analysis.